For HIV-1 to enter a cell, its envelope glycoprotein gp120 must sequentially associate with the cellular receptor CD4 and a chemokine co-receptor, principally CCR5 or CXCR4. CD4 induces in gp120 a conformational change necessary for a high affinity-association with co-receptor. The CD4 and co-receptor-binding sites are the most conserved and functionally important regions of gp120. The co-receptor-binding site includes at least two highly conserved sulfotyrosine-binding pockets which associate with sulfotyrosines in the CCR5 amino-terminus. These pockets are to some extent formed in the absence of CD4, and are conserved in both CCR5- and CXCR4-using viruses (R5 and X4 viruses, respectively). Association of gp120 with CCR5 sulfotyrosines may induce a second conformational change in gp120 necessary for its subsequent interaction with the remainder of the CCR5 ectodomain. Here we describe a novel peptide inhibitor of HIV-1 entry, formed of two components: a 27-amino-acid CD4 mimetic developed in the laboratory of the late Claudio Vita, and a 12-amino-acid tyrosine-sulfated CCR5 mimetic that we have developed. When the two mimetic peptides are linked, the combined double mimetic peptide has dramatically higher affinity for gp120 than either mimetic alone, and is a potent inhibitor of both R5 and X4 HIV-1 isolates. This potency likely derives from cooperativity between the CD4- and CCR5-mimetic components, and perhaps enhanced triggering of irreversible conformational changes in the HIV-1 envelope glycoprotein. An improved double-mimetic peptide has a number of potential advantages as a therapeutic, especially in concert with other entry inhibitors in late clinical trials or current use. We propose to improve and characterize this novel entry inhibitor, with focus on how it works in concert with enfuvirtide and small-molecule CCR5 antagonists. PUBLIC HEALTH RELEVANCE: In this proposal, we present a novel peptide inhibitor of HIV-1 entry that combines two peptides, each of which emulates an obligate HIV-1 receptor. This class of inhibitors has a number of potential advantages, especially in concert with other treatments for HIV-1 infection. We seek here to improve and characterize this inhibitor.